The goal of this continuing collaborative project is to test the abilities of specific IgA antibodies to provide immune protection against Cryptosporidium parvum in the intestine. We have generated a panel of 6 monoclonal, dimeric IgA antibodies from mucosally-derived hybridomas. They are partially characterized, showing distinct antigen specificities directed against surface components of C. parum, and all show passive immune protection in the infant mouse model. The project has 3 major aims; 1) To continue production and characterization of monoclonal IgAs. We will establish antigen and epitope specificities of the available monoclonal IgAs and identify possible cross-reactivity with antigens and surface components previously identified by others (including 15 and 47 kD antigens, GP900, and the putative lectin studies in Project 1). Additional IgA hybridomas will be generated after oral immunizations with recombinant protein fragments of a protective, glycosylated antigen GP900 (in collaboration with Drs. J. Crabb, ImmuCell Corp. and C. Petersen, UCSF). Epitope specificities will be compared with those of available monoclonal IgGs, polyclonal antisera, and bovine colostral antibodies. 2) To define in detail the capacities of specific monoclonal IgA antibodies to prevent or reverse C parvum infection. Protective capacities of individual IgAs to prevent C. parvum infection in vivo will be quantitatively compared in the infant mouse model. IgA antibodies with differing antigen specificities will be tested for synergistic protection. Parallel tests will be done in vitro suing MDBK and Caco-2 cell monolayers. Treatment of chronically-infected SCID mice with IgA hybridoma "backpack" tumors will demonstrate whether continuous secretion of protective IgAs can reverse established infection in intestine and biliary tree. using protective IgA monoclonals identified above we will test the ability of added IgA monoclonals identified above, we will test the ability of added IgA to enhance passive protection by hyperimmune bovine colostrum (from ImmuCell) in vivo and in vitro. 3). To investigate possible sites and mechanisms of IgA protection at epithelial surfaces. The distribution of antigens recognized by protective IgAs on C. parvum oocysts, sporozoites and intracellular forms during infection of epithelial cells will be analyzed by high LM immunofluorescence and EM immunogold labeling of intact organisms, sections of infected mucosa, and Caco-2 cell monolayers (defined in Project 2). Immunolabeling and ultrastructural analysis will also be used to elucidate the interactions among parasites, protective IgA antibodies, and epithelial cell surfaces during challenge with C. parvum in vivo and in vitro. The detailed evaluation of IgA antibodies proposed in this project will serve to establish the role of secretory IgA in normal mucosal protection against cryptosporidiosis, and could lead to more effective passive immune prophylaxis and immunotherapy in immunocompromised hosts.